Non-infective Stages Research Articles

Heteroleptic Oxidovanadium(V) Complexes with Activity against Infective and Non-Infective Stages of Trypanosoma cruzi.

Five heteroleptic compounds, [VVO(IN-2H)(L-H)], where L are 8-hydroxyquinoline derivatives and IN is a Schiff base ligand, were synthesized and characterized in both the solid and solution state. The compounds were evaluated on epimastigotes and trypomastigotes of Trypanosoma cruzi as well as on VERO cells, as a mammalian cell model. Compounds showed activity against trypomastigotes with IC50 values of 0.29–3.02 μM. IN ligand and the new [VVO2(IN-H)] complex showed negligible activity. The most active compound [VVO(IN-2H)(L2-H)], with L2 = 5-chloro-7-iodo-8-hydroxyquinoline, showed good selectivity towards the parasite and was selected to carry out further biological studies. Stability studies suggested a partial decomposition in solution. [VVO(IN-2H)(L2-H)] affects the infection potential of cell-derived trypomastigotes. Low total vanadium uptake by parasites and preferential accumulation in the soluble proteins fraction were determined. A trypanocide effect was observed when incubating epimastigotes with 10 × IC50 values of [VVO(IN-2H)(L2-H)] and the generation of ROS after treatments was suggested. Fluorescence competition measurements with DNA:ethidium bromide adduct showed a moderate DNA interaction of the complexes. In vivo toxicity study on C. elegans model showed no toxicity up to a 100 μM concentration of [VVO(IN-2H)(L2-H)]. This compound could be considered a prospective anti-T. cruzi agent that deserves further research.

Polyunsaturated fatty acid metabolites: biosynthesis in Leishmania and role in parasite/host interaction

Inside the human host, Leishmania infection starts with phagocytosis of infective promastigotes by macrophages. In order to survive, Leishmania has developed several strategies to manipulate macrophage functions. Among these strategies, Leishmania as a source of bioactive lipids has been poorly explored. Herein, we assessed the biosynthesis of polyunsaturated fatty acid metabolites by infective and noninfective stages of Leishmania and further explored the role of these metabolites in macrophage polarization. The concentration of docosahexaenoic acid metabolites, precursors of proresolving lipid mediators, was increased in the infective stage of the parasite compared with the noninfective stage, and cytochrome P450-like proteins were shown to be implicated in the biosynthesis of these metabolites. The treatment of macrophages with lipids extracted from the infective forms of the parasite led to M2 macrophage polarization and blocked the differentiation into the M1 phenotype induced by IFN-γ. In conclusion, Leishmania polyunsaturated fatty acid metabolites, produced by cytochrome P450-like protein activity, are implicated in parasite/host interactions by promoting the polarization of macrophages into a proresolving M2 phenotype.

Faecal avoidance differs between the sexes but not with nematode infection risk in mandrills

Animals have evolved a wide range of behaviours that act as barriers to decrease the risk of parasite infection. Faecal avoidance may, for example, limit contact with orofaecally transmitted parasites, such as gastrointestinal nematodes. When present in faeces, however, nematode eggs need to mature before reaching their infective stage. If strategies have evolved in hosts to specifically avoid nematodes, old faeces with infective larvae should elicit stronger avoidance behaviour than fresh faeces that contain noninfective stages. Here, we carried out two experiments to test the hypothesis that mandrills, Mandrillus sphinx, an Old-World primate, exhibit specific behavioural strategies to avoid nematode infection. Our results show that individuals did not avoid faeces in a nonfeeding context but did avoid eating food items contaminated with faecal material, females more so than males. However, neither the presence of nematodes nor the age of faeces influenced the level of avoidance observed, suggesting that mandrills avoid faecal material in general rather than nematodes specifically when foraging.

Defining host range: host-parasite compatibility during the non-infective phase of the parasite also matters.

Host range and parasite specificity determine key epidemiological, ecological and evolutionary aspects of host-parasite interactions. Parasites are usually classified as generalists or specialists based on the number of hosts they feed on. Yet, the requirements of the various stages of a parasite may influence the suitability of a given host species. Here, we investigate the generalist nature of three common ectoparasites (the dipteran Carnus hemapterus and two species of louse flies, Pseudolynchia canariensis and Ornithophila metallica), exploiting two avian host species (the European roller Coracias garrulus and the Rock pigeon Columba livia), that frequently occupy the same breeding sites. We explore the prevalence and abundance of both the infective and the puparial stages of the ectoparasites in both host species. Strong preferences of Pseudolynchia canariensis for pigeons and of Carnus hemapterus for rollers were found. Moderate prevalence of Ornithophila metallica was found in rollers but this louse fly avoided pigeons. In some cases, the infestation patterns observed for imagoes and puparia were consistent whereas in other cases host preferences inferred from imagoes differed from the ones suggested by puparia. We propose that the adult stages of these ectoparasites are more specialist than reported and that the requirements of non-infective stages can restrict the effective host range of some parasites.

Parasite-induced alteration of plastic response to predation threat: increased refuge use but lower food intake in Gammarus pulex infected with the acanothocephalan Pomphorhynchus laevis

Larvae of many trophically-transmitted parasites alter the behaviour of their intermediate host in ways that increase their probability of transmission to the next host in their life cycle. Before reaching a stage that is infective to the next host, parasite larvae may develop through several larval stages in the intermediate host that are not infective to the definitive host. Early predation at these stages results in parasite death, and it has recently been shown that non-infective larvae of some helminths decrease such risk by enhancing the anti-predator defences of the host, including decreased activity and increased sheltering. However, these behavioural changes may divert infected hosts from an optimal balance between survival and foraging (either seeking food or a mate). In this study, this hypothesis was tested using the intermediate host of the acanthocephalan parasite Pomphorhynchus laevis, the freshwater amphipod Gammarus pulex. We compared activity, refuge use, food foraging and food intake of hosts experimentally infected with the non-infective stage (acanthella), with that of uninfected gammarids. Behavioural assays were conducted in four situations varying in predation risk and in food accessibility. Acanthella-infected amphipods showed an increase in refuge use and a general reduction in activity and food intake. There was no effect of parasite intensity on these traits. Uninfected individuals showed plastic responses to water-borne cues from fish by adjusting refuge use, activity and food intake. They also foraged more when the food was placed outside the refuge. At the intra-individual level, refuge use and food intake were positively correlated in infected gammarids only. Overall, our findings suggest that uninfected gammarids exhibit risk-sensitive behaviour including increased food intake under predation risk, whereas gammarids infected with the non-infective larvae of P. laevis exhibit a lower motivation to feed, irrespective of predation risk and food accessibility.

Rapid and highly sensitive detection of single nematode via direct MALDI Mass Spectrometry

For the first time, we demonstrate the feasibility for the use of MALDI-TOF MS for rapid, direct and sensitive detection of single adult root-knot nematode, Meloidogyne incognita and their second stage juvenile (J2). We have proposed simple pretreatment protocols and have demonstrated that the crushed and washed nematodes yielded better spectra. We also report the differentiation between the harmless and harmful stages of the nematode based on mass spectrometric profiling. Peaks at m/z 4350, 4692, 4933, 8725 were only present in the adult stages, while m/z 3220, 3433, 3485, 3830, 6540, 7444, 7770 were unique to the J2 infective stage. The only common peak to both the phases was at m/z 3277. Thus, we show that MALDI-TOF MS can be used to differentiate between the infective and non-infective stage of the nematode and the detection sensitivity of MALDI-MS could be applied to a single nematode analysis.

PROTECTION FIRST THEN FACILITATION: A MANIPULATIVE PARASITE MODULATES THE VULNERABILITY TO PREDATION OF ITS INTERMEDIATE HOST ACCORDING TO ITS OWN DEVELOPMENTAL STAGE

Many trophically transmitted parasites with complex life cycles manipulate their intermediate host behavior in ways facilitating their transmission to final host by predation. This facilitation generally results from lowering host's antipredatory defenses when the parasite is infective to the final host. However, a recent theoretical model predicts that an optimal parasitic strategy would be to protect the intermediate host from predation when noninfective, before switching to facilitation when the infective stage is reached. We tested this hypothesis in the fish acanthocephalan parasite Pomphorhynchus laevis using the amphipod Gammarus pulex as intermediate host. Gammarids parasitized by noninfective stage of P. laevis (acanthella) hid significantly more under refuges than uninfected ones. In addition, acanthella-infected gammarids were less predated upon by trout than uninfected ones. As predicted, a switch toward decreased antipredatory behavior of G. pulex and enhanced vulnerability to predation was found when P. laevis reached the stage infective to its final host. The parasites appear to be able to exploit plasticity in host antipredatory responses, and shift the host optimal response toward their own optimal balance.

PCR detection of Dirofilaria immitis in Aedes aegypti and Culex pipiens from urban temperate Argentina

Dirofilariasis, a mosquito-borne disease of dogs caused by the nematode Dirofilaria immitis (Leidy; Spirurida: Onchocercidae), has now become a growing zoonotic concern. Based on direct microscopical observation, Aedes aegypti (L.) and Culex pipiens L. (Diptera: Culicidae) have been previously incriminated as potential vectors of D. immitis in urban temperate Argentina. In this study, an effort was made to provide evidence for this assumption by screening of mosquitoes for D. immitis infection using a polymerase chain reaction (PCR) assay. PCR primers were developed to specifically amplify the D. immitis-16S rRNA gene and to reliably detect 100th of the genomic equivalent (10 pg) of the infective third-stage larvae in mosquito pools of up to 30 individuals. Collection of mosquitoes was performed between September 2007 and April 2008 in premises known to be inhabited by D. immitis-infected dogs in Greater Buenos Aires. The final collection comprised 453 specimens belonging to 11 mosquito species of the genera Aedes, Culex, Ochlerotatus, and Psorophora. PCR assays were performed on 82 pools (n ≤ 20) of heads and abdomens separately, as this allows differentiating infective and non-infective stages of the parasite, respectively. Identification of the non-infective stage of D. immitis in A. aegypti and C. pipiens provided additional strong support of transmission of the parasite by these species. To our knowledge, this was the first PCR screening for D. immitis-infected mosquitoes in South America.

Alternative trans‐splicing of the Trypanosoma cruzi LYT1 gene transcript results in compartmental and functional switch for the encoded protein

Trypanosoma cruzi has a complex life cycle that includes infective and non-infective stages in distinct hosts. Control of gene expression by the parasite must adjust to rather diverse circumstances. Through stage-regulated, alternative trans-splicing of the primary transcript, the T. cruzi LYT1 gene generates two protein products differing in the presence or absence of 28 amino acids at their amino end. We find that the shorter protein, kLYT1, is located at two spots in the mitochondrial kinetoflagellar zone and its expression reverts the 'accelerated stage development' phenotype of the LYT1-null mutant. The larger product, mLYT1, localizes on the plasma membrane. The signal for membrane localization presents characteristics of a type II anchor including the possibility of cleavage. Expression of mLYT1 reverts the 'loss of virulence' phenotype associated to diminished haemolytic activity at acid pH, but stage development still progresses at an accelerated rate. This compartmentalization switch of LYT1 results in two surprisingly different functions: haemolytic activity at acid pH for mLYT1, and a putative involvement in mitochondrial metabolism for kLYT1. We conclude that alternative trans-splicing plays an important role in stage-regulated control of gene expression in trypanosomatids.

Contamination of Drinking Water Supplies in Mazandaran - Iran

Introduction : Direct relationship has been reported between the prevalence of some parasitic diseases and the presence of microorganisms in water. There are possible contamination of water resources, due to the climate changes and increasing the groundwater level. The purpose of this research was to determine the contamination rate of drinking water supplies to parasites in Mazandaran Province, north of Iran, during 2002-2003. Materials and Methods : In this cross-sectional study, 989 water samples which randomly taken based on towns population and number of primary health care centers from 12 cities of Mazandaran province. Collected water samples were transferred to the laboratory in sterile containers and standard condition. The samples were filtered and then analyzed according to the WHO guidelines. Direct method and lugol staining procedure were used to identify the parasites. In cases of cryptosporidium presence, centrifugal flotation techniques using Sheather's sugar solution and modified Ziehl-Neelsen staining method were performed. McMaster counting slides were used for parasites count in 0.3ml. Results: Almost 20% (197 out of 989) of water samples were contaminated with different parasites. 20 different types of parasites were identified in 197 contaminated samples, which 53 (26.9%) were pathogenic, 100 (50.8%) non-pathogenic, and 44 (22.3%) non-infective stages of parasites. Conclusions : Drinking water contamination to parasites has been reported about 15 percent in most of Mazandaran areas. Our findings confirmed increasing prevalence of intestinal parasitic infections in the other studies in this province. According to high prevalence of water contamination and its contributes to diseases, proper implementation of providing safe drinking water should be considered by water supply companies.

Development-related effects of an acanthocephalan parasite on pairing success of its intermediate host

The acanthocephalan parasite, Acanthocephalus dirus , undergoes larval development in the aquatic isopod, Caecidotea intermedius , before completing its life cycle in the intestine of a fish (e.g. sunfish). Inside C. intermedius , the parasite develops through noninfective acanthor and acanthella stages and into the cystacanth stage, which is infective to definitive hosts. Research has shown that individual A. dirus induce phenotypic changes in C. intermedius that increase conspicuousness to definitive hosts, and that the level of modification is most pronounced when the parasite has developed into the cystacanth stage. We examined whether development into the cystacanth stage also correlated with changes in mating behaviour of the host. Using a field survey, we quantified the pattern of development of larval A. dirus to identify specific periods when either infective (cystacanth) or noninfective (acanthor, acanthella) stages dominated. Males infected with cystacanth-stage larvae had lower pairing success than males infected with larvae in the noninfective stages. To identify the behavioural mechanisms underlying this relation (i.e. male response to females, female resistance), we used a field-based experiment to examine the relation between parasite development and mating interactions between males and females. Cystacanth-infected males were less likely to initiate mating attempts with females than were acanthor/acanthella-infected males, and this effect could explain the population-level mating pattern. We discuss the results in the context of both adaptive and nonadaptive mechanisms underlying parasite-mediated changes in mating behaviour.

Dirofilariasis in Argentina: Historical review and first report of Dirofilaria immitis in a natural mosquito population

Argentina is one of the four South American countries where the presence of Dirofilaria immitis is currently confirmed. The objective of this study was to review information on dirofilariasis in the country, and to report our recent findings on mosquito vectors. Since the first report of dogs with unidentified microfilariae in 1926, D. immitis was found in seven provinces and canine prevalence ranged 0–71% at local scale. National prevalence was 8% by the end of the 1980s and current information is available only for Buenos Aires Province. Four pulmonary human infections of D. immitis and one subcutaneous of Dirofilaria sp. were documented. The common coati was the only wild host found, and natural infection in mosquitoes was not previously reported in the country. In our recent mosquito survey in Greater Buenos Aires, we captured and dissected 2380 mosquitoes belonging to 20 species. According to a minimum temperature of 14 °C, the potential transmission period (PTP) for D. immitis in Buenos Aires covers 6 months, and the most favourable period (mean temperature above 20 °C) takes place from the middle of November to the beginning of April. To identify potential vectors of the parasite, we assessed weekly abundances of mosquito species during those PTP estimated previously. We found two specimens of Culex pipiens and one of Aedes aegypti carrying non-infective stages of D. immitis. These two highly anthropophilic mosquitoes may enhance the role of D. immitis as zoonotic agent in temperate Argentina.

Intra-specific host sharing in the manipulative parasite Acanthocephalus dirus: does conflict occur over host modification?

The acanthocephalan parasite Acanthocephalus dirus induces a colour change in the intermediate host, the aquatic isopod Caecidotea intermedius, which increases transmission to definitive hosts (creek chub, sunfish). We examined the potential for conflict to occur between infective (cystacanth) and non-infective (acanthor, acanthella) stages of A. dirus over the level of colour modification that should be induced when these stages share a host. Using a field survey, we showed that host sharing by infective and non-infective stages was relatively common and that infective and non-infective stages differed in their effects on colour modification. Non-infective stages induced a colour change over 40% of the body, whereas infective stages induced a colour change over 80%. Thus, conflict could occur between stages over the level of modification that should be induced. We then showed that mixed-stage infections induced a colour change in the host that was consistent with the level of modification induced by the infective stage. We discuss the potential significance of these results to patterns of host modification and their effects on stage-related survival in nature.

Trypanosoma cruzi: identification of a galactose-binding protein that binds to cell surface of human erythrocytes and is involved in cell invasion by the parasite

Trypanosoma cruzi must invade mammalian host cells to replicate and complete its life cycle. Almost all nucleated mammalian cells can be invaded by the parasite following a receptor–ligand recognition as an early prerequisite. In this work, we describe a 67-kDa lectin-like glycoprotein that binds to desialylated human erythrocyte membranes in a galactose-dependent way. This protein is present on the parasite surface in both infective and non-infective stages of T. cruzi. More interestingly, we demonstrate by lectin-immuno-histochemistry assays that the 67 kDa protein is involved in the recognition of host-cell receptors in mouse cardiac tissue and human cardiac aortic endothelium and mammary artery tissue. Moreover, antibodies against the 67 kDa glycoprotein inhibit in vitro host-cell invasion by 63%. These data suggest that the 67 kDa glycoprotein in vivo is needed for host-cell invasion by T. cruzi. Index Descriptors and Abbreviations: Trypanosoma cruzi ( T. cruzi), Red blood cell membranes, 67-kDa lectin-like glycoprotein (LLGP-67), Shed acute phase antigen (SAPA), Red blood cell ghosts (RBCG), Lythium diiodesalicylate (LIS), Total homogenate (TH), Phosphate buffered saline (PBS), Phosphate saline buffer, 0.01% Tween 20 (PBS-T), Phenylmethylsulphonyl fluoride (PMSF), Nα- p-Tosyl- l-lysinechloromethyl ketone (TLCK), Ultraviolet (UV), Concanavalin A (Con A), Peanut agglutinin (PNA), Tetramethylbenzidine (TMB), Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), Enzyme-linked immunosorbent assay (ELISA), Immunofluorescence asssay (IFA), Bovine serum albumin (BSA), Indirect hemagglutination assay (IHA), Dimethyl sulphoxide (DMSO).

Developmental modification of the lipophosphoglycan from Leishmania major promastigotes during metacyclogenesis

Lipophosphoglycan was isolated from the dividing, noninfective stage and from the nondividing metacyclic stage of Leishmania major promastigotes. The lipophosphoglycans were characterized by SDS-PAGE and by chromatographic and quantitative analysis of phosphatidylinositol-specific phospholipase C- and mild acid-generated fragments. The results revealed two stage-specific structural differences: (i) an increase in size of the metacyclic form of the glycoconjugate due to an approximate doubling in the number of its salient phosphorylated saccharide units; and concomitantly, (ii) a subtle compositional change in these units. Gas-liquid chromatographic analysis indicated that the phosphatidylinositol lipid anchor was developmentally conserved. These developmental modifications suggest important roles for the lipophosphoglycan which have not been previously considered, such as promoting complement resistance within the vertebrate host, and midgut attachment and release within the sand fly vector.

Occurrence of the Noninfective Stage of Ceratomyxa shasta in Mature Summer Chinook Salmon in the South Fork Salmon River, Idaho

Abstract This report documents the occurrence of Ceratomyxa shasta in mature summer chinook salmon (Oncorhynchus tshawytscha) in the South Fork Salmon River, Idaho, in 1984 and notes the parasite's possible contribution to prespawning mortalities in this stock of fish. This represents an extension of the range of the noninfective stage of this parasite.

The action of insect juvenile hormone on the hatching of eggs of the nematode, Haemonchus contortus, and its role in the development of infective and non-infective stages

1. 1. With a gas phase of CO 2-free air the inhibitory action of the analogues of juvenile hormone, “dichlorofarnesoate” and farnesol (JH) on the hatching of eggs of Haemonchus contortus was markedly affected by [H +]. The pK of the inhibitory effect was 6.8; above pH 7 inhibition was weak, below 6.5 it was strong. 2. 2. The inhibition of hatching of eggs in 0.1 M NaCl containing JH, 3 × 10 −4 M, and under a gas phase of air, increased as the temperature was raised from 20 to 37°C. 3. 3. In buffer at pH 6 and under air, increases in temperature to 37°C did not inhibit hatching in the absence of added JH. However, when CO 2 was removed from the gas phase hatching was inhibited at the higher temperatures. 4. 4. Acetazolamide, 10 −3M, and ethoxyzolamide, 2 × 10 −5 M, inhibited the exsheatment (ecdysis) of third stage juveniles of H. contortus. 5. 5. The significance of these results in providing a hypothesis for features in the life cycle of nematodes, and in providing a hypothesis describing the action of JH on the target cell, is discussed.

ELECTRON MICROSCOPY OF LATENT PSITTACOSIS VIRUS IN MCCOY CELLS.

Kajima, Masahiro (University of Notre Dame, Notre Dame, Ind.), Nehama Sharon, and Morris Pollard. Electron microscopy of latent psittacosis virus in McCoy cells. J. Bacteriol. 88:709-715. 1964.-Replication of psittacosis virus in McCoy cells was observed periodically by electron microscopy, and was correlated with the appearance of inclusion bodies in infected cells stained by acridine orange fluorochrome. Three well-defined morphological stages characterized the replication cycle: (i) noninfectious initial body, (ii) noninfectious intermediate body, and (iii) infectious mature or elementary body. When replication of virus was interrupted by aminopterin, the virus failed to develop beyond the noninfective initial body stage; however, after treatment of such cultures with folinic acid, intermediate and mature virus particles appeared in the cells. Latent psittacosis virus, as described here, represented a virus in which the replication process was interrupted at a noninfective (eclipse) stage of the developmental cycle.